Researchers frequently use 3T3-L1 adipocytes as a fat cell line, but the capacity of this line for insulin-mediated glucose transport is lower than that of primary isolated fat cells. In this study, we found that 5-azacytidine (5-aza-C), DNA methyltransferase 1 inhibitor, enhanced insulin-stimulated 2-deoxyglucose (2-DG) transport in 3T3-L1 cells after adipogenic differentiation. We next examined the expression of the genes related to glucose transport and insulin signal transduction. The insulin independent glucose transporter, glucose transporter 1 (GLUT1), showed lower expression in 5-aza-C pre-treated 3T3-L1 adipocytes, than in un-treated control adipocytes, while the expression of insulin dependent transporter GLUT4 remained unchanged. In addition, insulin receptor substrate-1 (IRS-1) was highly expressed in 5-aza-C pre-treated adipocytes. Based on DNA microarray and functional annotation analysis, we noticed that 5-aza-C pretreatment activated the tumor suppressor p53 pathway. We confirmed that in 5-aza-C adipocytes, p53 expression was markedly higher, and the methylation level of CpGs in its promoter region was lower than in un-treated control adipocytes. Moreover, pharmacological inhibition of p53 restored GLUT1 and IRS-1 expression to the same level as in un-treated 3T3-L1 adipocytes, and significantly decreased insulin-mediated 2-DG uptake. These results suggest that glucose transport capacity in adipocytes is influenced by DNA methylation status, and demethylation induced by 5-aza-C increased it possibly through the p53 signaling pathway.